Title:  Combination therapy method for treating chronic hepatitis B

United States Patent:  6,495,521

Issued:  December 17, 2002

Inventors:  Horwitz; David L. (Hillsborough, CA)

Assignee:  SciClone Pharmaceuticals, Inc. (San Mateo, CA)

Appl. No.:  764838

Filed:  January 17, 2001

The present invention is aimed at augmenting the success rate of using thymosin in treatment of chronic hepatitis B, by employing a combination therapy using thymosin with antiviral agents which are effective in inhibiting DNA synthesis or DNA polymerase during replication of the hepatitis B virus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating chronic hepatitis B infection in mammals comprising concurrently administering to chronic hepatitis B-infected subjects a therapeutically effective amount of at least one thymosin, and an inhibitorily effective amount of at least one hepatitis B virus replication or DNA polymerase inhibitor compound, either free or as a pharmaceutically acceptable salt, in a pharmaceutically acceptable vehicle, which results in improved or beneficially synergistic clinical effects in such subjects. These combination therapies are more effective than when each is administered as a sole treatment modality.

The term "thymosin" as used herein is intended to include any immunopotentiating polypeptide naturally occurring in the thymus gland or produced by chemical or recombinant means, or fragments derived from any of these polypeptides. "Thymosin" includes, thymosin Fraction Five (TF-5), thymosin alpha-1 and any biologically active peptide fragment (such as C-terminal 4-28 and 15-28, and N-terminal 1-8, 1-14 and 1-20 fragments), analog or derivative of any of those. As used herein, the term "thymosin alpha-1" is intended to refer to the 28-mer described below, with or without the N-acetyl group, as well as biologically active analogs of the sequence (i.e. deletion, substitution and addition mutants), which are substantially homologous to the peptide sequence shown below.

Thymosin Fraction Five (TF-5), originally described by Goldstein et al. (Proc. Nat'l Acad. Sci. (USA), 69:1800-1803 (1972)), is a partially purified extract of bovine thymus containing at least 40 peptide components, 20 of which have been purified to homogeneity or near homogeneity; it contains about 0.6% of thymosin alpha-1. Low, et al., "Thymosins: Structure, Function and Therapeutic Application", Thymus, 6:27-42 (1984), incorporated by reference.

A peptide that is "substantially homologous" to thymosin alpha-1 is one in which at least about 30%, preferably at least about 85% to about 90% and most preferably about 95%, of the amino acids match over a defined length of the molecule, with the sequence depicted below.

A "biologically active" fragment or analog of thymosin or thymosin alpha-1, is a fragment or analog of thymosin or thymosin alpha-1, respectively which retains a significant amount of the activity of the native molecule, i.e., which is capable of decreasing serum HBV DNA and/or hepatitis B surface antigen, as described further below.

The term "treatment" or "treating" as used herein refers to either (i) the prevention of infection or reinfection (prophylaxis) or (ii) the reduction or elimination of indicators of chronic hepatitis B.

A "therapeutically effective amount" of thymosin is an amount of the peptide which has the capability of changing the measurable parameters of hepatitis B infection. The parameters that will normally be monitored are serum hepatitis B surface antigen and serum viral DNA. Response is defined as a significant decrease of either of these parameters. An amount of peptide which has the ability of eliciting a response is considered a "therapeutically effective amount." HBV DNA can be monitored using the spot hybridization assay described in Mutchnick, M. G. et al., Hepatology (1991) 14:409-415 and Lieberman, H. M. et al., Hepatology (1983) 3:285-291, both of which are incorporated by reference herein. Alternatively, the presence of HBV DNA in the blood can be measured using standard PCR technology. See, e.g. U.S. Pat. Nos. 4,683,202 and 4,683,195, incorporated herein by reference in their entirety. HBV DNA can also be detected through use of a commercially available kit from Abbott Laboratories, North Chicago, Ill. Serum hepatitis B surface antigen levels can be monitored using standard RIAs, as described by Mutchnick, M. G. et al., Hepatology (1991) 14: 409-415 incorporated herein by reference, or by standard ELISAs.

The present invention relates to a combination therapy using a medicament containing as active ingredient at least one thymosin, either in free form or in the form of a pharmaceutically acceptable salt. The thymosin may be administered alone or mixed with a pharmaceutically acceptable vehicle or excipient.

A most preferred embodiment of the present invention is to use a medicament containing as the thymosin active ingredient, thymosin alpha-1 for the treatment of chronic hepatitis B infection. The native molecule is a 28-mer, having the amino acid sequence shown below:

Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu- Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu- Val-Val-Glu-Glu-Ala-Glu-Asn-OH. (Seq ID No: 1

Thymosin alpha-1, as well as fragments and analogs thereof are easily synthesized using standard methods of peptide synthesis, known to those of skill in the art. U.S. Pat. Nos. 4,148,788 and 4,855,407 describe the solution phase and solid phase synthesis, respectively, of thymosin alpha-1, and are incorporated herein by reference in their entirety. See also, Young, J. D., Solid Phase Peptide Synthesis, 2nd ed. (Pierce Chemical Company 1984); and Barany, G. and Merrifield, R. B., The Peptides: Analysis, Synthesis, Biology, Vol. 2 (Gross, E. and Meienhofer, J. eds., Academic Press 1980), for a discussion of solid phase peptide synthesis; and Bodansky, M. Principles of Peptide Synthesis (Springer-Verlag 1984); and The Peptides: Analysis, Synthesis, Biology, Vol. 1 (Gross, E. and Meienhofer, J. eds., Academic Press 1980), for solution phase peptide synthesis.

Thymosin alpha-1 can also be isolated directly from appropriate tissue expressing thymosin alpha-1, using techniques readily known in the art. This is generally accomplished by first preparing a crude tissue extract which lacks cellular components and several extraneous proteins. The thymosin alpha-1 can be further purified, i.e. by column chromatography, HPLC, immunoadsorbent techniques or other conventional methods well known in the art. U.S. Pat. No. 4,079,127 discloses a method for purifying thymosin alpha-1 from calf thymus and is incorporated herein by reference in its entirety.

Thymosin alpha-1 and fragments or analogs thereof, can also be produced recombinantly using methods well known to those of skill in the art. See, e.g. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press 1989); Oligonucleotide Synthesis (M. J. Gait ed. 1984).

Thymosins can also be obtained from commercial sources (e.g. Alpha 1 Biomedicals, Inc., Foster City, Calif.).

Typically, the thymosin compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles. The active ingredient can be mixed with vehicles containing excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent to those skilled in the art. See, e.g. Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th edition, (1975). The composition or formulation to be administered will, in any event, contain a quantity of the peptide adequate to reduce or eliminate HBV DNA and/or HBsAg from the serum of the subject being treated.

Thymosins may be administered orally or parenterally. Parenteral administration may be achieved either intravenously, subcutaneously, or by intramuscular injection. Injectable formulations will contain an effective amount of the active ingredient in a vehicle, the exact amount being readily determined by one skilled in the art. The active ingredient may range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate. The quantity to be administered depends on factors such as the age, weight, health, severity of the condition, duration of treatment required of the subject to be treated, and the other drugs of the combination of the invention that are being concurrently administered.

In the most preferred embodiment of the present formulations of the present invention, between about 300 .mu.g to about 3000 .mu.g, preferably between about 900 .mu.g to about 1200 .mu.g of thymosin alpha-1 per square meter of body area will be administered. Such dosages can be given once a week up to once a day, preferably two to three times a week for a treatment course of between two months to three years, preferably for about 6 to 12 months. A preferred dosage unit form for pharmaceutical use is 1.6 mg of lyophilized thymosin alpha-1 per vial, and this material is reconstituted prior to use by the addition of diluent. Other effective dosages can be readily established by one of ordinary skill in the art without undue experimentation through routine dose response trials.

Additional formulations which are suitable for other modes of administration include suppositories and in some cases, aerosol, intranasal, oral formulations, and sustained release formulations. For suppositories, the vehicle composition will include traditional binders and carriers, such as, polyalkaline glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%. Oral vehicles include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like. These oral compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.

Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.

Controlled or sustained release formulations are made by incorporating the peptide into carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel.RTM. copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures. The peptides can also be presented using implanted mini-pumps, well known in the art.

Furthermore, the peptides may be formulated into compositions in either neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active peptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as for example, sodium, potassium ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

An inhibitorily effective amount of at least one antiviral agent, particularly hepatitis B virus replication or DNA polymerase inhibitor compound is included in the combination chemotherapy regimen of the present invention. The infectious virion of hepatitis B contains a small, circular DNA molecule that is partly single-stranded and a DNA polymerase that can make the DNA fully double-stranded. Its mechanism of replication involves an RNA intermediate. The antiviral agents which act to inhibit hepatitis B viral replication or inhibit DNA polymerase activity of the present invention comprise purine or pyrimidine nucleoside analogs.

An "inhibitorily effective amount" of an antiviral drug or agent is an amount of the drug which inhibits HBV virus replication, measured by a decrease in viral DNA in the blood, as measured by PCR or other method known in the art.

Antiviral agents of the present invention which are pyrimidine nucleoside analogs include ddI, ddC, AZT and FIAU (fluoro-iodo-arabinofuranosyl-uracil) (see Table below). Antiviral agents of the present invention which are purine nucleoside analogs include acyclovir, ribavirin, ganciclovir, and vidarabine (see Table below). AZT, ddC, ddI and FIAU act as polynucleotide chain terminators. Similarly, acyclovir and other purine analogs act as polynucleotide chain terminators. These analogs act as faulty substrates, thus preventing DNA transcription. The mode of action of ribavirin is most likely interference with viral mRNA, resulting in inhibition of viral replication.

The antiviral agents of the present invention, are given in an appropriate pharmaceutical dosage formulation. The pyrimidine nucleoside analogs of the present invention can be given intravenously or orally to chronic hepatitis B-infected subjects at effective viral inhibiting dosages and according to regimens appropriate to the severity of the disease and clinical factors. However, when given in combination with a thymosin, a lower daily dosage for a subject can be devised according to the clinical parameters and tests listed below. Those with skill in the art will, without undue experimentation, be able to devise dosages depending on the clinical condition of patients and the parameters discussed below.

Further, the thymosin and antiviral agent are administered concurrently in that the treatment administration of each drug overlap in time. Preferably, the antiviral agent is administered first with the administration of thymosin beginning at the same time or within four weeks after the first administration of the antiviral agent. Most preferably, administration of thymosin is begun within one week after administration of the antiviral agent has begun.

The following Table lists various antiviral agents of use in the invention with exemplary modes of action and exemplary dosages and modes of administration.

Antiviral agents are known and can be chemically synthesized or obtained commercially. For example: AZT, acyclovir and trifluridine (Burroughs Wellcome Co., Research Triangle Park, N.C.); ganciclovir (Syntex, Palo Alto, Calif.); vidarabine (Parke-Davis, Morris Plains, N.J.); idoxuridine (Smith Kline Beecham Pharmaceuticals, Philadelphia, Pa.); foscarnet (Astra Pharmaceuticals, Westborough, Mass.); amantadine (DuPont Pharmaceuticals (Wilmington, Del.)); rimantadine (Forest Pharmaceuticals, Maryland Heights, Mo.); ribavirin (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.); didanosine (Bristol Myers Squibb Company, Evansville, Ind.); zalcitabine (Roche Products, Nutley, N.J.).

In a preferred protocol, administration of a pyrimidine or purine nucleoside analog (e.g., AZT, ddI, ddC, FIAU, acyclovir, ribavirin) at a dosage level and manner described in the Table is begun with thymosin alpha-1 at a dosage level of 1.6 mg. subcutaneously one to four times weekly, most preferably twice weekly. Antiviral therapy is continued until viral DNA levels are negative and thymosin alpha-1 is continued for an additional three months.

Measurement of severity of the disease can be accomplished in subjects. Such measurements or markers of chronic hepatitis B include the level of the enzymes ALT (alanine aminotransferase, sometimes referred to as SGPT) and AST (aspartate aminotransferase, sometimes referred to as SGOT) in the blood. These methods and techniques are standard in this art.

Claim 1 of 15 Claims

What is claimed is:

1. A method for treating chronic hepatitis B infection in mammals comprising concurrently administering to a subject having chronic hepatitis B a therapeutically effective amount of at least one thymosin, and an inhibitorily effective amount of at least one purine nucleoside analog which is a hepatitis B viruc replication or DNA polymerase inhibitor compound, either free or as pharmaceutically acceptable salt, is a pharmaceutically acceptable vehicle.
 

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